Hepatocellular carcinoma (HCC, also referred to as malignant hepatoma) is a primary malignancy of the liver. HCC most commonly appears in patients with chronic viral hepatitis (e.g. hepatitis B) and/or cirrhosis with any etiology. Non-alcoholic steatohepatitis (NASH) has also been found to be a risk factor for the development of HCC. Examples of other risk factors include high aflatoxins exposure and iron overload conditions such as hemochromatosis. In some cases, cryptogenic HCC is developed in patients with no history of liver disease or known risk factors.
HCC incidence worldwide is constantly increasing. In some parts of the world, such as in sub-Saharan Africa, China, Hong-Kong and Taiwan, HCC is a major health problem, probably due to high exposure to hepatitis viruses like B and C and to regional exposure to environmental pathogens.
The pathogenesis of HCC is not completely known, however mitochondrial dysfunction is suggested to be involved. HCC cells show genetic and metabolic alterations with a decreased mitochondrial function (Warburg O (1956). “On the origin of cancer cells”. Science 123 (3191): 309-14).
Treatment of HCC may be directed towards a cure, or focused on relief of symptoms and prolongation of life. The type of treatment is typically selected according to the tumor size and stage. When the tumor is small (less than 2-3 cm), limited to one lobe of the liver, without evidence of invasion of the liver vasculature and in a well preserved liver function, surgical resection may be performed. Other treatment options include liver transplantation, radiofrequency ablation (RFA) and transarterial chemoembolization (TACE) (for treatment algorithms, see, for example, the Barcelona Clinic Liver Cancer (BCLC) Staging System). In RFA, radiofrequency energy is transmitted to the tumor via a needle electrode which is advanced into the tumor under image guidance (such as X-ray screening, CT scan or ultrasound). The radiofrequency waves passing through the needle lead to tumor destruction by thermal coagulation and protein denaturation. In TACE, chemotherapy is administered directly to the tumor via a catheter, and blood supply to the tumor is cut-off. In addition to the procedures described above, oral medicines may also be administered. For example, sorafenib tosylate (Nexavar™), an oral medicine that blocks tumor growth by activating the intrinsic mitochondrial pathway (Kurosu, “Sorafenib induces apoptosis specifically in cells expressing BCR/ABL by inhibiting its kinase activity to activate the intrinsic mitochondrial pathway”, Cancer Research 2009 May 1; 69(9):3927-36.), is approved for patients with advanced HCC.
In general, small or slow growing tumors may be successfully treated if diagnosed early. However, early diagnosis is difficult, partially because most of the patients who develop HCC have no symptoms other than those related to their longstanding liver disease. Surveillance of high-risk groups, such as cirrhotic patients, is usually performed to facilitate early detection of HCC (see, for example, AASLD Guidelines).
The detection and diagnosis of HCC is typically based on imaging tests, serology tests and sometimes biopsy. Imaging tests include, for example, abdominal ultrasound, helical computed tomography (CT) scan, triple phase CT scan and magnetic resonance imaging (MRI). Serology tests include measurement of blood levels of alpha-fetoprotein (AFP), where high levels of AFP are associated with HCC. The typical strategy is 6-monthly surveillance with AFP and ultrasound.
Assessment of HCC is also performed as part of post-treatment monitoring. Evaluation of treatment efficacy and determination of active vs. inactive HCC are usually performed radiologically using contrast-enhanced CT or MRI. Exemplary contrast media is Lipiodol®, an iodized oily agent that is selectively retained within the tumor microvessels. Lack of vascular enhancement in the treated lesion is typically indicative of positive response to the treatment. AFP is not accurate enough as a follow-up tool, and the monitoring of AFP levels after therapy does not replace imaging. The ideal imaging interval is unknown, but initially a 3-4 month interval is commonly used to monitor HCC lesions after initial treatment.
The standard techniques for HCC diagnosis and follow-up have several drawbacks. For example, imaging methods based on CT or MRI, are considered expensive, must be performed in a hospital by a skilled practitioner, and associated with high radiation and side effects such as contrast-media-induced nephropathy in the case of CT/MRI. In addition, methods such as AFP measurements are insufficiently sensitive or specific.
Breath tests based on monitoring 13CO2 levels, which is a by-product of metabolism of 13C-labeled substrates by the liver, have been proposed as a tool for evaluation of liver function. If the hepatic metabolism of a test compound results in the formation of carbon dioxide, and the appropriate carbon is labeled, the exhalation of labeled CO2 (which is measurable, for example, in mass spectrometry or non-dispersive infrared analyzer), reflects the hepatic clearance of the original labeled compound and may be used to asses specific liver functions. For example, compounds metabolized by hepatocytes cytochrome P450 enzymes may be used in the assessment of liver microsomal function. Exemplary compound is methacetin. As another example, compounds that undergo metabolism in liver mitochondria may be used in the assessment of liver mitochondria function and may be used to detect certain liver conditions (see for example, Grattagliano et al. (2010) Eur J Clin Invest, 40 (9): 843-850 and Portincasa et al. (2010) Clujul Medical, 83: 23-26).
One exemplary molecule is ketoisocaproate (KICA), a compound that undergoes decarboxylation in liver mitochondria. Use of KICA breath tests in evaluation of liver function in HCC patients has been reported (Palmieri et al. (2009) Journal of Surgical Research 157, 199-207). In this study, the effect of two different HCC treatments on liver function was evaluated. Cirrhotic patients with and without HCC were tested using, inter alia, KICA decarboxylation. At baseline, patients with HCC had significantly lower 13C-KICA breath test values compared with healthy controls and cirrhotic patients without HCC. Minor but significant changes in 13C-KICA breath test values emerged between cirrhotic patients without HCC and healthy controls (lower values were observed for the cirrhotic patients without HCC compared to healthy control). The patients were treated by either TACE or RFA, after which 13C-KICA levels were again tested at day 1, day 30 and day 180. The authors summarized: “ketoisocaproate decarboxylation was unaffected by TACE but decreased after RFA (−27%, P<0.05)”, and concluded: “RFA not TACE appears to spare residual (microsomal) liver mass, but induces such a transient stunning effect on mitochondrial function”.
Another exemplary molecule that is metabolized by liver mitochondria is Octanoate. Octanoate is a medium chain fatty acid that enters mitochondria and undergoes β-oxidation generating acetyl coenzyme A (acetyl-CoA). Acetyl-CoA enters the Krebs cycle and is oxidized to CO2 unless it is utilized for the synthesis of other energy-rich compounds.
WO 2007/054940, to the Applicant of the present invention, discloses breath test devices and methods for the evaluation of liver functional and metabolic capacity or to assess liver health and/or degree of liver injury. For example, a method of evaluating a liver condition is disclosed, the method includes on-line monitoring a metabolic product of octanoic acid, a salt or a derivative of octanoic acid, in a subject's breath after administering to the subject isotope labeled octanoic acid, a salt or a derivative thereof. As another example, a device for evaluating a liver condition is disclosed, the device includes one or more sensors adapted to monitor on-line an isotope level of a metabolic product of labeled octanoic acid, or a salt or a derivative thereof in a subject's breath and a controller adapted to sample measurements of the one or more sensors at a continuous mode. The method and device may be used in distinguishing between a non-alcoholic fatty liver and non-alcoholic steatohepatitis conditions in a subject.
WO 2010/013235, to the Applicant of the present invention, discloses breath test devices and methods that may be used for the evaluation of liver functional and metabolic capacity or to assess liver health and/or degree of liver injury. For example, a method of detecting abnormal beta-oxidation associated with insulin resistance or alcoholic liver disease or non-alcoholic fatty liver disease or metabolic syndrome is disclosed, the method includes monitoring a metabolic product of octanoic acid, a salt or a derivative of octanoic acid, in a subject's breath after administering to the subject isotope labeled octanoic acid, a salt or a derivative thereof.
The complexity of liver metabolism imposes challenges on assessing its function, and therefore interpretation of breath test results is not always straightforward. Examples of potential limitations for the interpretation of breath test results include the presence of confounding variables (e.g. exercise), the typology of gastric emptying kinetics, the hepatic first pass metabolism of the administered substrates, and the presence of competing pathways of elimination and metabolism of compounds. Additional factors that should be taken into account include, for example, the presence of mitochondrial metabolism occurring in organs other than the liver (extra hepatic metabolism, when hepatic metabolism is of interest, would negatively impact test relevance), possible dilution of exogenous labeled compound in a larger pool of unlabelled compound, and endogenous production of unlabelled CO2 which can vary substantially from subject to subject. Furthermore, there are many factors that result in high intra- and inter-patient variability, and different disease etiologies may impact different functions of the liver and may result in a different breath test outcomes.
There still remains a need for cost effective, accurate and simple methods and systems for surveillance, detection, prognosis and monitoring response to treatment of hepatocellular carcinoma.